Hybridization between complementary nucleic acids is an implicit feature in the Watson-Crick model for DNA structure that is exploited for many applications of the biological and biomedical arts. For example, virtually all methods for replicating and/or amplifying nucleic acid molecules are initiated by a step in which a complementary oligonucleotide (typically referred to as a “primer”) hybridizes to some portion of a “target” nucleic acid molecule. A polymerase then synthesizes a complementary nucleic acid from the primer, using the target nucleic acid as a “template.” See Kleppe et al., 1971 J. Mol. Biol. 56: 341-61.
One particular application, known as the polymerase chain reaction, PCR, is widely used in a variety of biological and medical arts. For a description, see Saiki et al., 1985, Science 230: 1350-54. In PCR, two or more primers are used that hybridize to separate regions of a target nucleic acid and its complementary sequence. The sample is then subjected to multiple cycles of heating and cooling, repeatedly hybridizing and dissociating the complementary strands so that multiple replications of the target nucleic acid and its complement are performed. As a result, even very small initial quantities of a target nucleic acid may be enormously increased, or “amplified,” for subsequent uses (e.g., for detection, sequencing, etc.).
Multiplex PCR is a particular version of PCR in which several different primers are used to amplify and detect a plurality of different nucleic acids in a sample—usually ten to a hundred or more different target nucleic acids. Thus, the technique allows a user to amplify and evaluate large numbers of different nucleic acids simultaneously in a single sample. The enormous benefits of high throughput, speed and efficiency offered by this technique has made multiplex PCR increasingly popular. However, achievement of successful multiplex PCR usually involves empirical testing as existing computer programs that pick and/or design PCR primers have errors. In multiplex PCR, the errors become additive and therefore good results are seldom achieved without some amount of trial and error. See Markouatos et al., 2002, J. Clin. Lab Anal. 16(1): 47-51; Henegarin et al., 1997, Biotechniques 23(3): 504-11.
Some applications using probes and primers are designed to distinguish between two or more sequences that differ by one or more nucleotides, such as assays designed for single nucleotide polymorphism (SNP) detection. In these assays, mutations of clinical significance differ by a single nucleotide from the wild-type sequence.
Stability and melting temperature, Tm, of nucleic acid duplexes is a key design parameter for a variety of applications utilizing DNA and RNA oligonucleotides (Petersen and Wengel, 2003, Trends Biotechnol. 21: 74-81; You et al., 2006, Nucleic Acids Res., 34: e60). The successful implementation of all techniques involving nucleic acid hybridization (including the exemplary techniques described, supra) is dependent upon the use of nucleic acid probes and primers that specifically hybridize with complementary nucleic acids of interest while, at the same time, avoiding non-specific hybridization with other nucleic acid molecules that may be present. For a review, see Wetmur, 1991, Critical Reviews in Biochemistry and Molecular Biology 26: 227-59. These properties are even more critical in techniques, such as multiplex PCR and microarray hybridization, where a plurality of different probes or primers is used, each of which may be specific for a different target nucleic acid.
Various modifications are available that can significantly affect the Tm of a nucleic acid duplex. The modifications can be placed at a terminal end, such as a minor groove binder (MGB) (Kutyavin et al., 2000, Nucleic Acids Research, 28(2): 655-61). The modifications can be placed on the backbone of the oligonucleotide, examples of which include phosphorothioates, phosphorodithioates and phosphonoacetates. The modifications can be located on the sugar moiety, examples of which include locked nucleic acids (LNAs), 2′-O-methyls, 2′-methoxyethylriboses (MOE's), ENA's (ethylene bicyclic nucleic acids). The modification can be located on the base moiety, examples of which include 5-methyl-dC and propynyl-dU and propynyl-dC.
LNAs are RNA modifications wherein a methyl bridge connects the 2′-oxygen and the 4′-carbon, locking the ribose in an A-form conformation, providing synthetic oligonucleotides with unique properties (Koshkin et al., 1998, Tetrahedron 54: 3607-30; U.S. Pat. No. 6,268,490). LNA modifications increase the stability of nucleic acid duplexes and the specificity of oligonucleotide binding to complementary sequences, e.g., genomic DNAs (Petersen and Wengel, 2003). Therefore, oligonucleotides containing LNA modifications may be used to improve accuracy and sensitivity of various biological applications and assays, e.g., antisense oligonucleotides, nucleic acid microarrays, sequencing, PCR primers, PCR probes and medical diagnostics.
Preliminary work has been performed to develop thermodynamic parameters for DNA duplexes containing an LNA modification (see McTigue et al., 2004, Biochemistry 43(18): 5388-05). McTigue et al. improved upon the older model of Tm prediction simply based upon the number of LNA additions and described sequence-dependent thermodynamic parameters for duplex formations containing a single LNA modification.
Since duplexes containing a single LNA analog represent only a fraction of LNA-containing duplexes, there is a need for sequence-dependent thermodynamic parameters for duplexes containing multiple LNA analogs, especially those containing multiple adjacent LNA analogs. The present invention includes methods to predict the stability and Tm of chimeric duplexes containing various amounts of locked nucleic acid modifications in oligonucleotide strands. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.